Biomedical subjects

Jan Matthes

Publications and source records attributed to Jan Matthes.

9 recordsLinked to original sources

PubMed2006

Mechanisms of [Ca2+]i transient decrease in cardiomyopathy of db/db type 2 diabetic mice.

Cardiovascular disease is the leading cause of death in the diabetic population. However, molecular mechanisms underlying diabetic cardiomyopathy remain unclear. We analyzed Ca2+-induced Ca2+ release and excitation-contraction coupling in db/db obese type 2 diabetic mice and their control littermates. Echocardiography showed a systolic dysfunction in db/db mice. Two-photon microscopy identified intracellular calcium concentration ([Ca2+]i) transient decrease in cardiomyocytes within the whole heart, which was also found in isolated myocytes by confocal microscopy. Global [Ca2+]i transients are constituted of individual Ca2+ sparks. Ca2+ sparks in db/db cardiomyocytes were less frequent than in +/+ myocytes, partly because of a depression in sarcoplasmic reticulum Ca2+ load but also because of a reduced expression of ryanodine receptor Ca2+ channels (RyRs), revealed by [3H]ryanodine binding assay. Ca2+ efflux through Na+/Ca2+ exchanger was increased in db/db myocytes. Calcium current, I(Ca), triggers sarcoplasmic reticulum Ca2+ release and is also involved in sarcoplasmic reticulum Ca2+ refilling. Macroscopic I(Ca) was reduced in db/db cells, but single Ca2+ channel activity was similar, suggesting that diabetic myocytes express fewer functional Ca2+ channels, which was confirmed by Western blots. These results demonstrate that db/db mice show depressed cardiac function, at least in part, because of a general reduction in the membrane permeability to Ca2+. As less Ca2+ enters the cell through I(Ca), less Ca2+ is released through RyRs.

Animals↗

PubMed2004

An aqueous extract of the marine sponge Ectyoplasia ferox stimulates L-type Ca2+-current by direct interaction with the Cav1.2 subunit.

Marine organisms have attracted much attention as a source of pharmacological tools or potential drugs. We have produced and screened a library of sponge extracts in search of biologically active compounds that may contain useful pharmaceutical lead structures. Sponges were collected from various locations and their aqueous extracts were freeze dried. Murine right and left atria were used to screen 75 extracts for putative cardiac effects. Among seven extracts with a positive inotropic and chronotropic effect the extract C47 from Ectyoplasia ferox proved to be the most active and was chosen for further analysis. C47 also produced a beta-adrenoceptor-independent, propranolol-resistant positive inotropic effect in human atrial trabeculae. To elucidate one possible mode of action the effects of C47 on L-type Ca(2+) current (I(Ca,L)) were measured with a standard patch-clamp technique. In isolated human atrial myocytes exposure to C47 increased peak amplitude of I(Ca,L) in a concentration-dependent manner. The threshold concentration was 15 microg/ml. In addition, voltage dependency of activation and steady-state inactivation were shifted to more negative potentials. C47 slowed the initial phase of time-dependent current inactivation and the recovery from inactivation. In cell-attached patches of HEK 293 cells expressing human Ca(v)1.2 addition of C47 to the bath solution did not affect gating properties, whereas inclusion of the extract into the pipette solution strongly increased single-channel activity, suggesting a direct effect on the pore-forming channel subunit. Despite its robust effect on I(Ca,L) C47 enhanced cardiac force of contraction by only a fraction of the maximum increase caused by high extracellular concentrations of Ca(2+) and failed to increase vascular tone. These findings suggest that the effect of C47 is restricted to the Ca(2+) channel.

Action Potentials↗

PubMed2004

Disturbed atrio-ventricular conduction and normal contractile function in isolated hearts from Cav1.3-knockout mice.

Cardiac L-type calcium channels are formed by two alpha-subunits, Cav1.2 (alpha(1C)) and Cav1.3 (alpha(1D)). In contrast to the uniform expression pattern of Cav1.2, Cav1.3 is highly expressed in sino-atrial node (SAN) and atrial tissue, but not in the ventricle. Accordingly, knockout of Cav1.3 (Cav1.3(-/-)) in mice was shown to lead to a cardiac phenotype characterised by severe bradycardia in vivo and in isolated SAN cells. Cav1.3 may therefore constitute a novel pharmacological target for specific bradycardic agents. RNAse protection assays of murine wild type hearts revealed rather high Cav1.3 levels comparable to Cav1.2, suggesting functional relevance of Cav1.3 outside specialised tissues such as SAN. Due to the lack of specific Cav1.3 blockers, we directly examined the functional role of Cav1.3 using isolated working hearts from adult wild type (WT) and Cav1.3(-/-) mice. Histological analysis of hearts revealed no pathological changes. Ventricular contractility and inotropic effects of isoproterenol were unaltered in Cav1.3(-/-) hearts. Severe sinus bradycardia already noted in vivo was accompanied by ventricular extrasystoles. This phenotype was restored to nearly normal values by the cumulative addition of isoproterenol. Electrocardiograms of Cav1.3(-/-) hearts revealed delayed atrio-ventricular (AV) conduction and a decoupling of heart rate and PR interval duration. Isoproterenol did not improve disturbance of AV conduction. In conclusion, suppression of Cav1.3 does not alter ventricular contractile function, and the decrease in sinus node frequency is counterbalanced by adrenergic stimulation. Importantly, bradyarrhythmia is partly due to an intrinsic AV node dysfunction, which is resistant to adrenergic counterbalance. These findings help to predict the clinical pattern of selective Cav1.3 blockade.

Adrenergic beta-Agonists↗

PubMed2004

Calcium channel function and regulation in beta 1- and beta 2-adrenoceptor transgenic mice.

Cardiac effects of catecholamines on the L-type calcium channel depend on beta-adrenoceptor subtype (beta(1)- vs. beta(2)-adrenoceptor). Chronic overexpression of these receptors leads to hypertrophy and early death at moderate (beta(1)) or excessive (beta(2)) levels of overexpression respectively. In order to examine the role of L-type calcium channels in altered cardiomyocyte calcium homeostasis found with beta(1)-adrenoceptor overexpression, and to understand the quantitative differences between beta-adrenoceptor subtypes regarding calcium channel regulation, we examined single channels in myocytes obtained from beta(1)- and beta(2)-adrenoceptor transgenic mice. The effects of the agonist isoproterenol were investigated and compared with acute receptor stimulation in the respective non-transgenic littermates. Channels from beta(1)-adrenoceptor transgenic mice have normal baseline activity, and channel number is not reduced. This contrasts to previous findings with beta(2)-adrenoceptor transgenic mice, where channel activity is depressed. Isoproterenol is unable to stimulate channel activity in both transgenic models. In conclusion, the L-type calcium channel is not likely to be involved in alterations of calcium handling of beta(1)-adrenoceptor transgenic myocytes. Furthermore, chronic beta(1)-adrenoceptor overexpression does not depress channel activity, giving another example of the difference between beta(1)- and beta(2)-adrenoceptor signal transduction.

Adrenergic beta-Agonists↗

PubMed2004

Single-channel gating and regulation of human L-type calcium channels in cardiomyocytes of transgenic mice.

Overexpression of human cardiac L-type Ca(2+) channel pores (hCa(v)1.2) in mice causes heart failure. Earlier studies showed Ca(v)1.2-mRNA increase by 2.8-fold, but whole-cell current density enhancement by </=1.5-fold only. Three possible explanations were examined: (1) poor translation of hCa(v)1.2 and of its accessory subunits, (2) altered sarcolemmal insertion of functional channels, and (3) lower single-channel activity of overexpressed channels. Western blots revealed a 2.7-fold increase of Ca(v)1.2 protein in transgenic myocytes, but less enhanced expression of beta(1a) and beta(1b) subunits. beta(2) and alpha(2)/delta were significantly lowered. Density of functional channels was increased by 3.0-fold. Single-channel gating was impaired in transgenic cardiomyocytes: open probability and ensemble average currents were reduced by 60%. Furthermore, channels of transgenic myocytes were not stimulated by 8-Br-cAMP, in contrast to wild-types. Expression of malcomposed, dysfunctional L-type Ca(2+) channels in murine cardiomyocytes overexpressing hCa(v)1.2 explains the moderate enhancement of whole-cell currents and illustrates compensatory mechanisms in a transgenic disease model.

Animals↗

PubMed2004

Ca2+-dependent modulation of single human cardiac L-type calcium channels by the calcineurin inhibitor cyclosporine.

OBJECTIVE: Activity of single L-type calcium channels (LTCC) is enhanced in human failing myocardium (Circulation 98 (1998) 969.), most likely due to impaired dephosphorylation. Protein phosphatase 2B (calcineurin) has recently been shown to be involved in heart failure pathophysiology. We now focus on the regulation of single LTCC by calcineurin that were prevented by Ca(2+)-free experimental conditions in our previous study. METHODS: Single LTCC currents were recorded in myocytes from human atrium and ventricle. Charge carriers were 70 mM Ba(2+), or a mixture of 30 mM Ca(2+) and 60 mM Ba(2+) to facilitate Ca(2+) permeation through recorded channels. The calcineurin inhibitor cyclosporine (10 microM) was used to reveal a putative role for calcineurin in regulation of LTCC. RESULTS: A mixture of Ca(2+) and Ba(2+) as charge carriers allowed for Ca(2+) permeation through recombinant human embryonic kidney cells and native (atrial and ventricular) human cardiac LTCC. With only Ba(2+) as the charge carrier, activities of both ventricular and atrial LTCC were strongly decreased by cyclosporine. In contrast, channel activity remained constant when Ca(2+) permeation was provided. In the presence of thapsigargin and (S)-BayK 8644, cyclosporine here even increased channel activity. CONCLUSIONS: We propose a dual cyclosporine effect on human cardiac LTCC. A non-specific inhibitory effect prevails with Ba(2+) permeation but can be compensated or overcome by a specific Ca(2+)-dependent stimulation with Ca(2+) permeation. More complete restoration of physiological Ca(2+) movements (e.g., Ca(2+) release from sarcoplasmic reticulum) will help to define even more precisely the involvement of calcineurin in regulation of human cardiac LTCC.

Barium↗

PubMed2003

Cardioprotection specific for the G protein Gi2 in chronic adrenergic signaling through beta 2-adrenoceptors.

Two subtypes of beta-adrenoceptors, beta 1 and beta 2, mediate cardiac catecholamine effects. These two types differ qualitatively, e.g., regarding G protein coupling and calcium channel stimulation. Transgenic mice overexpressing human beta 2-adrenoceptors survive high-expression levels, unlike mice overexpressing beta 1-adrenoceptors. We examined the role of inhibitory Gi proteins, known to be activated by beta 2- but not beta 1-adrenoceptors, on the chronic effects of human beta 2-adrenoreceptor overexpression in transgenic mice. These mice were crossbred with mice where G alpha i2, a functionally important cardiac Gi alpha-subunit, was inactivated by targeted gene deletion. Survival of beta 2-adrenoreceptor transgenic mice was reduced by heterozygous inactivation of G alpha i2. Homozygous knockout/beta 2-adrenoreceptor transgenic mice died within 4 days after birth. Heterozygous knockout/beta 2-adrenoreceptor transgenic mice developed more pronounced cardiac hypertrophy and earlier heart failure compared with beta 2-adrenoreceptor transgenic mice. Single calcium-channel activity was strongly suppressed in heterozygous knockout/beta 2-adrenoreceptor transgenic mice. In cardiomyocytes from these mice, pertussis toxin treatment in vitro fully restored channel activity and enhanced channel activity in cells from homozygous G alpha i2 knockout animals. Cardiac G alpha i3 protein was increased in all G alpha i2 knockout mouse strains. Our results demonstrate that G alpha i2 takes an essential protective part in chronic signaling of overexpressed beta 2-adrenoceptors, leading to prolonged survival and delayed cardiac pathology. However, reduction of calcium-channel activity by beta 2-adrenoreceptor overexpression is due to a different pertussis-toxin-sensitive pathway, most likely by G alpha i3. This result indicates that subtype-specific signaling of beta 2-adrenoreceptor functionally bifurcates at the level of Gi, leading to different effects depending on the G alpha isoform.

Animals↗

PubMed2002

The influence of tutor qualification on the process and outcome of learning in a problem-based course of basic medical pharmacology.

Limited access to expert tutors is a problem that can be addressed by using tutors from different stages of medical or non-medical (under-, post-) graduate education. To address whether such differences in qualification affect the results of process evaluation by participants or their learning outcome (exam results), we analysed the data of a 4-year prospective study performed with 787 3rd-year medical students (111 groups of 5-10 participants) taking an obligatory problem-based learning (PbL)-course of basic pharmacology. We compared peer tutors (undergraduate medical students, >/=4th year), non-expert (junior) staff tutors (physicians, pharmacists, veterinarians, biologists, or chemists during postgraduate education), and expert (senior) staff tutors (completed postgraduate education). Evaluation scores related to PbL gave the highest values for senior staff-led groups. The tutor's performance score of peer-led groups did not differ from those of staff-led groups, but the score obtained from groups tutored by junior staff was lower than that obtained with senior staff tutors. Students' weekly preparation time tended to be lower in peer-led groups, while learning time spent specifically on exam preparation seemed to be increased compared to PbL-groups of staff tutors. As a putative confounding variable, tutors' experience in coaching PbL-groups was also investigated. Groups led by experienced tutors, defined as tutors with at least one term of previous PbL tutoring, were found to have significantly higher evaluation scores. Interestingly, neither tutors' subject-matter expertise (peer students, junior staff, or senior staff) nor their teaching-method expertise showed any influence on PbL-groups' mean test scores in a written exam. This indicates that the effect of tutor expertise on the learning process is not associated with a difference in learning outcome when just factual knowledge is assessed by traditional methods.

Analysis of Variance↗

PubMed2002

Single-channel pharmacology of mibefradil in human native T-type and recombinant Ca(v)3.2 calcium channels.

To study the molecular pharmacology of low-voltage-activated calcium channels in biophysical detail, human medullary thyroid carcinoma (hMTC) cells were investigated using the single-channel technique. These cells had been reported to express T-type whole-cell currents and a Ca(v)3.2 (or alpha 1H) channel subunit. We observed two types of single-channel activity that were easily distinguished based on single-channel conductance, voltage dependence of activation, time course of inactivation, rapid gating kinetics, and the response to the calcium agonist (S)-Bay K 8644. Type II channels had biophysical properties (activation, inactivation, conductance) typical for high-voltage-activated calcium channels. They were markedly stimulated by 1 microM (S)-Bay K 8644, allowing to identify them as L-type channels. The channel termed type I is a low-voltage-activated, small-conductance (7.2 pS) channel that inactivates rapidly and is not modulated by (S)-Bay K 8644. Type I channels are therefore classified as T-type channels. They were strongly inhibited by 10 microM mibefradil. Mibefradil block was caused by changes in two gating parameters: a pronounced reduction in fraction of active sweeps and a slight shortening of the open-state duration. Single recombinant low-voltage-activated T-type calcium channels were studied in comparison, using human embryonic kidney 293 cells overexpressing the pore-forming Ca(v)3.2 subunit. Along all criteria examined (mechanisms of block, extent of block), recombinant Ca(v)3.2 interact with mibefradil in the same way as their native counterparts expressed in hMTC cells. In conclusion, the pharmacologic phenotype of these native human T-type channels--as probed by mibefradil--is similar to recombinant human Ca(v)3.2.

Calcium Channel Blockers↗